1. Field of the Invention
The invention relates to the identification and isolation of a G protein-coupled receptor cloned from rat taste buds and functionally expressed in Chinese hamster ovary (CHO) cells. The receptor couples negatively to a cAMP cascade, and has been identified as a taste receptor for glutamate.
2. Description of Related Art
Chemoreceptor cells in taste buds monitor the chemical environment of the oral cavity and generate signals that lead to taste perceptions. Taste transduction for simple salts involves altered permeation of the cell membranes of receptors by ions such as Na+, K+ or H+.1 The resulting receptor currents in taste bud cells stimulate neurotransmitter release to excite sensory afferents, ultimately leading to perceptions such as xe2x80x98saltyxe2x80x99 or xe2x80x98sourxe2x80x99. Taste transduction for larger organic molecules, such as sugars, amino acids or a heterogeneous collection of compounds that elicit perception of bitterness, is thought to include binding at specific receptors on the taste cell plasma membrane.1,2 
Some of the proteins that orchestrate this plethora of sensory transduction mechanisms have been identified using molecular biological methods. Taste receptor cells express G proteins, including xcex1-gustducin3, a-transducin4, a number of additional Gxcex1 subunits5,6, several Gxcex2 subunits and a taste-specific Gxcex37. Phosphodiesterases8 and a cyclic nucleotide-gated channel8 cloned from mammalian taste buds could potentially participate in sensory transduction pathways. Epithelial sodium channels demonstrated in taste buds presumably underlie xe2x80x98saltyxe2x80x99 and xe2x80x98sourxe2x80x99 transduction9-11. Detectable receptor activity for xe2x80x98bitterxe2x80x99 stimuli is found in membrane preparations from taste tissue12. Although a number of candidate taste-G protein-coupled receptors (GPCRs) have been proposed13-15, their functional significance in taste transduction has not been established2.
Sweet, sour, salty, bitter, discussed hereinabove, and umami constitute basic taste qualities. Umami denotes the taste of the glutamate moiety in monosodium L-glutamate (L-MSG), a compound that occurs naturally in protein-rich and other foods. Taste transduction for glutamate is hypothesized to entail stimulation of neurotransmitter-like ionotropic and metabotropic glutamate receptors16-18. A number of ionotropic glutamate receptors are expressed in lingual tissue, although none seems preferentially localized to taste buds17. Metabotropic glutamate receptors (mGluR1-8) constitute a family of GPCRs that are found in many neuronal cells19. In taste receptor cells, molecular, physiological and behavioral evidence implicates a metabotropic receptor similar or identical to mGluR4 in taste transduction for L-glutamate20. Such evidence includes the findings that mGluR4 is expressed in rat taste buds17,21 and that an mGluR4-selective ligand, L-AP4, mimics the taste of L-MSG in conditioned taste aversion in rats17 and in human psychophysical measurements 22. Further, both L-MSG and L-AP4 interact synergistically with nucleotide monophosphates to elicit umami taste responses23,24. Additionally, stimulating taste buds with glutamate decreases cellular cAMP46 and alters membrane conductances25, a signaling cascade also triggered by mGluR4. Collectively, these findings are consistent with the transduction of L-glutamate taste by an mGluR4-like receptor. Nevertheless, several lines of evidence indicate apparent discrepancies between umami taste and the properties of mGluR4. The concentrations of glutamate needed to elicit taste and to activate the neurotransmitter receptor mGluR4 differ markedly. The detection threshold for L-MSG in recordings from sensory afferents is 0.1-0.3 mM in juvenile and 1-3 mM in adult rodents26,27, whereas mGluR4 requires glutamate in the micromolar range. Further, the ability of glutamate agonists to stimulate mGluR4 does not correlate fully with their umami taste28. Additionally, umami taste does not seem to be blocked by a known antagonist of mGluR423. These observations suggest that the receptors transducing umami taste may differ significantly from mGluR4, particularly in the glutamate-binding domain.
The glutamate-binding domain of the mGluR is contained within the large extracellular N terminus. Although detailed structural information is lacking, a model of the N terminus of mGluRs is based on the structure of a bacterial periplasmic leucine-isoleucine-valine binding protein (LIVBP)29. Experimental verification of this model includes mutation of contacting amino acids29, expression of truncated extracellular domains that retain binding characteristics30 and chimeric receptors with distinct agonist sensitivities31. Thus, the extracellular N terminus might be a plausible site for differences between neurotransmitter and taste receptors for glutamate.
Thus, while sensory transduction for many taste stimuli such as sugars, some bitter compounds and amino acids is thought to be mediated via G protein-coupled receptors (GPCRs), no such receptors that respond to taste stimuli have heretofore been identified. Monosodium L-glutamate (L-MSG), a natural component of many foods, is an important gustatory stimulus believed to signal dietary protein. L-MSG, which conveys the umami taste, is often employed by industry to overcome or mask the bitterness associated with protein hydrolyzates used in food and health care products. Therefore, a need exists to identify taste receptors responsible for umami taste sensations in order to better understand the physiological and biochemical mechanisms behind such taste sensations. Identification of the umami taste receptor and its functional properties would permit, inter alia, food scientists tailor ligands to enhance or mimic the umami taste perception via this receptor.
The invention relates to the identification and isolation of a GPCR cloned from mammalian taste buds and functionally expressed in CHO cells. The receptor couples negatively to a cAMP cascade and shows an unusual concentration-response relationship. The similarity of its properties to MSG taste suggests that this receptor is a glutamate receptor responsible for the umami taste sensation.
A preferred embodiment of the invention is a rat metabotropic glutamate taste receptor having a molecular weight of approximately 68 kDa, encoded by the amino acid sequence of SEQ ID NO:13. The receptor functions as a umami taste receptor. The receptor exhibits a truncated extracellular N-terminal domain comprising about 50% fewer amino acids than are present in neurotransmitter mGluR4 metabotropic glutamate receptors.
Another preferred embodiment is an isolated mRNA molecule encoding a rat metabotropic glutamate taste receptor, the mRNA having the nucleic acid sequence of SEQ ID NO:1.
Another preferred embodiment is an isolated mRNA molecule encoding a metabotropic glutamate taste receptor comprising a subsequence of nucleic acid sequence of SEQ ID No.:14 having at least 85% sequence identity to SEQ ID NO.:1.
Yet another embodiment is a mammalian cell transfected with cDNA encoding a rat metabotropic glutamate taste receptor of SEQ ID NO.: 13, wherein the cDNA expressed in the mammalian cell is capable of being translated into immunologically recognizable metabotropic glutamate taste receptors. Preferably, the mammalian cell is a Chinese hamster ovary cell. The expressed metabotropic glutamate taste receptors in the Chinese hamster ovary cell exhibit an EC50 of about 250 to about 300 xcexcM glutamate.
Still another preferred embodiment of the invention is a method of screening samples for umami mimicking compounds comprising transfecting mammalian cells with cDNA encoding metabotropic glutamate taste receptor mRNA, the mRNA comprising the nucleic acid sequence of SEQ ID NO:1. The transfected cells are cultured in an environment that promotes expression of immunologically recognizable metabotropic glutamate taste receptors, and are then treated with a compound that induces cAMP production. The cells are incubated with a sample containing a potential umami mimicking compound capable of binding to the metabotropic glutamate taste receptors. Introduction of the sample containing the potential umami mimicking compound occurs simultaneously with introduction of the compound that induces cAMP. After exposure to such a sample, the amount of cAMP produced is measured. Thereafter, suppression of cAMP production is correlated with umami mimicking compound binding to the metabotropic glutamate taste receptors. Preferably, the transfected cells are Chinese hamster ovary cells. Preferably, the compound that induces cAMP production comprises forskolin.